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We studied antimutagenic properties of bioginseng, a biotechnological product obtained from the callus culture of the ginseng root cells by alcohol extraction (bioginseng-1) and lyophilization (bioginseng-2). We have estimated the frequency of sister-chromatid exchange and chromosome aberrations in the culture of Chinese hamster cells, of chromosome aberrations in cells of the Ehrlich-ICF ascite strain, and of micronuclei in the mouse bone marrow cells. The bioginseng exerted an antimutagenic effect with respect to nitrosomethylurea and cyclophosphamide. The sister-chromatid change in the culture of Chinese hamster cells decreased under the influence of bioginseng due, apparently, to enhanced DNA repair. The most distinct protective effect was observed when ginseng was introduced 2 h prior to the cell treatment with mutagens.

The ultrastructural organization of some chromosome regions (telomere, centromere, nucleolus organizer region), which can be identified undoubtedly under electron microscope after in situ fixation, was studied using serial ultrathin sections of pig embryo kidney cells. The ultrastructure of these regions was compared with patterns of their differential staining revealed by C-method and by fluorochromes, chromomycin A3 and DAPI, specific for GC- and AT-rich base pair regions. It is shown that telomere regions of chromosome are organized primarily by 20 nm fibrils of DNP, which form a clear chromonema--a thread of chromatin with thickness nearly 100 nm. In the centromere regions of metacentric chromosomes, fibrils 20 nm in diameter predominate, while in several acrocentric chromosomes--fibrils 10 nm thick are prevailing. It was suggested that the observed differences may depend on the DNA composition in these regions. The nucleolus organizer regions contain the fibrillar material 5-11 nm in diameter.

The effect of a 20% Hanks [correction of Hanx] solution on living cells of pig embryo kidney culture induces sharp changes in the structural and functional organization of nucleoli. 10-15 min after the beginning of the effect, no nucleoli are observed under the light microscope. The ultrathin sections reveal only fibrillar centers (FC) and a dense fibrillar component. Under these conditions nucleoli do not incorporate 3H-uridine. However, 30-120 min after the cells are transferred from the hypotonic to the normal isotonic medium, the nucleoli cannot be distinguished from initial ones by their form, size, location in the nucleus, ultrastructure and the 3H-uridine labelling pattern. This indicates that the effect of a 20% Hanks [correction of Hanx] solution on nucleoli of living PE cells is reversible. It has been suggested that reconstruction of nucleoli in the full culture medium may proceed at the expense of both synthesis de novo and the material of initial nucleoli.

The karyotypic structure of the Indian muntjak skin fibroblast cell line, with 7 chromosomes in modal class, was compared in the cells cultures, with 10 and 3% of fetal calf serum of two different lots. In addition, the effect of the same serum lot on chromosome variability in the two different Indian muntjac skin fibroblast sublines differing in the modal class stability was investigated. In both the cases, the serum affects the cell distribution for the chromosome number. The analysis of correlations between the main and other structure variants of the karyotype (MSVK and SVK) showed that the karyotypic variants are selected for the balanced genotype rather than for particular chromosomes. The changed cultural conditions resulted primary in the selection of the preexisting SVK to convert into MSVK. The lowering in the serum content to 3% induced the increase in chromosome aberrations. The frequency of chromosome aberrations depends on both the serum lot and the chromosomal composition of sublines. Chromosomal breaks and dicentrics are the predominant types of chromosomal disturbances. Dicentrics are produced mainly by chromosomes 1 and 2. The data obtained may suggest that sera could affect karyological characteristics of cultured cells. This indicates that the cytogenetical monitoring is necessary during a long-term cultivation of cell lines.

The dynamics of some karyotype characteristics throughout the spontaneous neoplastic evolution of embryonal fibroblasts of different mouse strains was studied using a differential staining of chromosomes. The independent variability of chromosome modal numbers, the number of polyploid cells in the cell line, the number of chromosomal rearrangements within one cell, the activity of formation of different types of chromosomal rearrangements and the activity of interactions of the homologous chromosomes in 15 studied cell lines were revealed. Possible levels of variability regulations of different karyotype characteristics are discussed.

Monoclonal antibodies (MAs) were produced against glucose-6-phosphate dehydrogenase (G6PD) of two vole species--Microtus arvalis and M. subarvalis. The binding level of the MAs to G6PD in both species were almost the same, which suggested that these MAs may be specific for the antigenic determinants common to G6PD of these species. The MAs produced against the vole G6PD were used for its intracellular localization. The patterns obtained after staining cells with the use of MAs against G6PD were the same as those obtained after staining with the use of antibodies against F-actin. There was a good conformity between the results of light and electron microscopic immunoenzyme analyses with regard to the binding of MAs produced to the actin microfilaments. It is concluded that G6PD is closely associated with actin microfilaments of the cell cytoskeleton.

We investigated effects of hypotonic treatment of cultured HL-60 cells and human fibroblasts, and of DRB on the quantity of AgNORs revealed following AgNO3 staining. The average number of grains increases after KCl hypotonic treatment. The lower the concentration of KCl in the solution, the higher the number of silver grains revealed in the nucleoli due to their dissociation into substructures. The use of adenosine analogue (DRB) before the hypotonic treatment of cells facilitates the following count of silver deposits. A maximum increase in the mean number of silver grains was observed after a 1.5 h incubation.

Karyotypic variability was investigated for the Chinese hamster lung cell line V-79, infected (contaminated) with a mycoplasma Acholeplasma laidlawii A. The mycoplasmal contamination did not affect cell distribution for the chromosome number. However, 30-70 days following cell culture contamination the increase in chromosomal aberrations was observed in the contaminated cell line primarily at the expense of chromosomal breaks. The following cyprofloxacin treatment of the culture resulted in mycoplasmal elimination and decrease in the frequency of chromosomal aberrations up to the control level. The analysis of G-banded chromosomes showed no significant differences in karyotypes of originally non-infected cells and cells after decontamination. The karyotypic variability, induced by mycoplasmal infection in V-79 cell line, differed from that in the muntjac skin fibroblast cell line, the latter being described elsewhere. A predominant type of chromosomal variability in V-79 cell line are chromosomal breaks, whereas in the muntjac fibroblast cell line dicentrics (telomeric fusions) were primarily observed, possible explanations of these differences being discussed. This may be presumably due to differences in karyotypic structure of these two cell lines, and to their different adaptation to culture conditions.

A comparative study has been made of the state of nucleoli in mononuclear cells and in cells with micronuclei formed around individual chromosomes under the action of low ionic strength solutions on living cells. The mean number of nucleoli in cells with micronuclei exceeds that in mononuclear interphase cells but is comparable with the number of nucleolus organizing regions (NORs) in metaphase. This argues for a supposition that 3-D separation of NO-chromosomes, obviously, does not lead to an activation of additional NORs as compared with mononuclear cells. Impregnation of micronuclei with AgNO3 reveals Ag-proteins in all the micronuclei irrespective of the degree of their maturity and the presence of NORs. Antibodies to B23 protein stain only mature micronuclei, irrespective of the presence of ribosomal genes in them. Ultrathin sections of micronucleoli manifest all the structural components of the normal nucleoli.

Original modification of the harvest procedure, hypotonic treatment and slide-making techniques were used to obtain prometaphase spreads of good quality fitted to G-banding or to FISH. Human blood cultures were synchronized with a methotrexate block during synthesis, and following this with a thymidine release. Cells were then proceeded into prometaphase and early metaphase and were quickly harvested without exposure to colcemid. Following incubation in a mixture of 0.56% KCl and 1% sodium citrate (1:1) for 20-23 min at room temperature, cells were fixed in 4 changes of fixative made of 3 parts of absolute methanol and 1 part of glacial acetic acid. Three or four 100-105 microliters volume of a suspension of fixed cells were blew with a strength through pasteur pipette tip from a distance 5-6 cm onto chilled superclean slides wetted by water. These slides were then quickly flame dried. Prior to staining, prometaphase bearing spreads were held in thermostat overnight at 37 degrees C. Prometaphases were G-banded with the aid of a modified technique elaborated in the Department of Clinical Genetics, Lund University Hospital (Sweden). The slides were incubated for 1-4 min at room temperature in 0.01 versene solution, containing 0.011% w/v trypsin (Difco), 0.4 mg/ml D-glucose, 0.17 mg/ml KCl, 1.7 mg/ml NaCl. Following a 15 sec incubation in a solution containing 1 mg/ml D-glucose, 0.4 mg/ml KCl, 8 mg/ml NaCl, G-banding was completed by staining the slides with 2% Giemsa (Merck) in pH 6.8 phosphate buffer for 4-5 min.(ABSTRACT TRUNCATED AT 250 WORDS)

In the process of hepatocarcinogenesis induced by dipin and partial hepatectomy in mice, initial hepatocytes are gradually replaced with a population of newly formed hepatocytes which originate from oval cells (Radaeva, Factor, 1990). It has been shown that the increase in hepatocarcinogenesis duration (2-11 weeks) along with the intensification of oval-cell reaction are accompanied by a progressive injury of cell genome in the initial hepatocyte population. This injury manifests itself by an accumulation of cells with micronuclei as well as by the development of high levels of polyploidy and aneuploidy. In the initial parenchyma, clastogenic and aneuploidizing effects of dipin are maximally pronounced at the stage of appearing hepatocyte nodules which consist of newly formed hepatocytes (8-11 weeks). By this time the proliferative pool comprises 83-92%, the amount of aberrant cells increases in average from 7 to 50%, and the indices of average nuclear ploidy in population enhance 6-8 times as compared with the average level of polyploidy in normal parenchyma. The quota of binuclear cells without micronuclei abruptly falls while the quota of binuclear cells with micronuclei and nuclear bridges increases reaching 3/4 of the total amount of binuclear cells. Binuclear cells with nuclear bridges comprise about one third of binuclear cells with micronuclei. At the stage of hepatocytic nodules (8-11 weeks after induction), the population of newly formed hepatocytes is characterized by the absence of morphologically damaged cells and of cells with micronuclei, a low incidence of binuclear cells (1-3%), and a high value of proliferative pool (83-96%).

A tumor promoter phorbol 12-myristate 13-acetate (PMA) induces characteristic reversible changes in the cell shape in certain fibroblastic lines. This reaction to PMA may be regarded as a prototype of reorganizations involving formation of stable cytoplasmic processes. Two specific drugs, Taxol and Colcemid, were used to study the role of microtubules and vimentin-containing intermediate filaments (IF) in the development of PMA-induced reorganizations. A short (I h) exposure to PMA induced formation of processes in the control cells rather than in the Colcemid treated cells having depolymerized microtubules and the IF that collapsed around the nucleus. A longer (3-4 h) exposure to PMA of the colcemid-treated cells induced a partial reversal of the IF collapse; those parts of peripheral lamellae that contained IF were transformed into narrow noncontractile processes. It is suggested that the local interaction of the IF with the actin system is an essential step in the formation of processes from lamellae.

One of the spindle poles of mitotic PK cells was irradiated with UV microbeam in metaphase or in anaphase. Electron microscopy showed that immediately after irradiation the microtubules around the centrosome were maintained, and that the ultrastructure of both irradiated and nonirradiated poles was similar. After microirradiation of the centrosome in metaphase, the mitotic halo around this centrosome was retained, but in due time the number of microtubules was getting less compared to that around the nonirradiated centrosome. When daughter cells with irradiated centrosomes are passing into the interphase, their centrioles are not separated from each other, no primary cilia are formed, and no replication of centrioles occurs. In the interphase cells with irradiated centrosomes, satellites are formed on the active centriole, but centrosome-attached microtubules are practically absent.

The karyotypic variability has been studied in a low chromosomal cell line of the Indian muntjak skin fibroblasts contaminated with Mycoplasma arginini R-16 and Acholeplasma laidlawii A. The mycoplasmal contamination exerted influence on the cell distribution for the chromosome number. The frequency of the modal class cells with 7 chromosomes, having the main structure variant of the karyotype (MSVK) 2 + 2 + 1 + 1 + 1, was seen to decrease, while the frequency of the submodal class cells with 6 chromosomes, having MSVK 2 + 2 + 1 + 1 because of the loss of Y1 chromosome from MSVK 2 + 2 + 1 + 1, to increase. The frequency of chromosomal aberrations, predominantly that of dicentrics, was higher in the contaminated variants. Dicentrics were mainly produced by chromosomes 1 and 2. They were formed by a nonrandom combination of chromosomes and their arms. Possible correlations are discussed between aneuploidy and dicentric chromosome formation in low chromosomal cell lines, and cell adaptations to culture conditions.

A method has been developed for preparing essentially pure primary cultures of neurons from 10-12 week old fetal human brain. This method is based on the 1) capacity of neurons to form multicellular aggregates, 2) cultivation in chemically defined medium, and 3) treatment with cytosine arabinoside as antimitotic agent. This procedure allowed the preparation of highly purified (95 per cent and more) neuronal aggregate cultures. Ultrastructural analysis of these cultures following one and 8 days has revealed their partial differentiation during cultivation.

The localization of DNA within nucleoli of mammalian cells was studied in human diploid fibroblasts (strain MRC-5), stained with chromomycin A3--the fluorochrome specific for G-C DNA base pairs. To change the rate of rDNA transcription, actinomycin D (0.08 mkg/ml) was added to log phase cells for 30-90 min. The stained cells were analysed with a highly sensitive video camera and deblurring algorithm (Jordan, Rawlins, 1990). The pattern of staining was compared with the ultrastructural peculiarities of the cells fixed in the same conditions. As the result, it is inferred that the intranucleolar DNA (rDNA) is consistent with the fibrillar centres as well as with the dense fibrillar component. The localization of rDNA may alter due to the transcriptional activity of ribosomal genes. The data obtained support the idea on the transcription of ribosomal genes inside the dense fibrillar component of the nucleolus.

A cytogenetic observation, that the sister chromatid exchanges (SCE) occur 3 times more frequently in a special form of xeroderma pigmentosum--XPII than in the norm, prompted a study of DNA replication in this rare disease. Using DNA fiber autoradiography, the rate of fork movement and the frequency of initiation in the adjacent clusters of replicons were estimated. The rate of fork movement was significantly slower than that in classical XP and in normal cells. Here evidence was provided on another defect in DNA replication in XPII that involves a significantly decreased number of simultaneously operating adjacent clusters of replicons, which results in a decreased rate of DNA chain-growth. According to the Painter replication model for SCE, the exchanges arise due to double-strand DNA breaks occurring on the border between two adjacent clusters, respectively, completely and partially replicated. A retarded fork-displacement rate together with a decreased rate of DNA-chain growth may cause this situation to persist longer than in the norm. Thus, our data provide a further support of the replication model for SCE. A similar combination of cytogenetic and molecular defects has been obtained earlier in the Bloom syndrome cells.

It has been shown that after enucleation of the PE cells with cytochalasin D the centrioles remain in approximately 80% of cytoplasts. Some cytoplasts contain only single centriole, either a mother (active) of a daughter (inactive) one. 20% cytoplasts have no centrioles. 2h after enucleation the centrosome structure in the cytoplasts did not differ from that in normal cells. 14-16 h after enucleation in many cytoplasts large secondary lysosomes and lipid droplets appeared around the centrosome. At this time in some cytoplasts in the centrosome we observed free microtubule convergence foci. 14-16 h after the enucleation, some cytoplasts have doubling centrioles. Under the influence of ouabain (30 min), the number of active centrioles oriented perpendicularly to the substrate plane in the cytoplasts increased. We suggest that the preferentially perpendicular orientation of centrioles to the substrate plane does not depend on the nuclear activity.

The influence of natural and synthetic polyamines, phalloidin, cytochalasin D, vinblastine, colchicine, puromycin, chlorpromazine, urea, glutaraldehyde, and ethanol on the phagosome-lysosome fusion and the content of F-actin in murine peritoneal macrophages has been studied. Fluorescent phallotoxin FITC-phalloidin was used to stain F-actin. Natural polyamines (spermine, spermidine, putrescine), phalloidin, ethanol (0.1 M) stimulated the phagosome-lysosome fusion and increased the mid-content of F-actin in macrophages. Cytochalasin D, vinblastine, colchicine, puromycin, chlorpromazine, urea, glutaraldehyde, ethanol (0.15 and 0.2 M) inhibited this process and decreased the mid-content of F-actin. Possible mechanisms of the interconnection of cytoskeleton and the phagosome-lysosome fusion are discussed.

Heterotypic cells in combined monolayer sheets can push each other from the substratum due to a competition in attachment of pseudopodia of contacting heterotypic cells: different types of epithelium can push each other and fibroblasts from the substratum in mixed cultures. The formation of extracellular matrix in mixed cultures was studied with antisera to fibronectin and laminin by immunofluorescent microscopy. It was shown that one group of cells in the sheet pushed another group together with their extracellular matrices. It is supposed that the interaction of contacting heterotypic cells and associated extracellular matrices may play an important role in distribution of different cell types in embryogenesis and carcinogenesis.

A 3D reconstruction of the centrosome region was made based on series of semithick sections in tissue culture cells. It was shown that: 1) the total number of microtubules attached to the centrosome is about 30-50 of which only 20% or less run farther than 2 microns away from the centrosome; 2) a certain number of short microtubules (less than 1 micron length) is present in the vicinity of the centrosome, the majority of them are attached to the centrosome; 3) many microtubules around the centrosome have no direct contact with either centrioles, or other microtubule-convergent structures; 4) the majority of free microtubules are comparatively long (more than 1 micron length); 5) almost all the microtubules running closer than 2 microns to the centrosome are oriented towards it with their proximal ends. The radial distribution of free microtubules around the centrosome support the supposition that they may appear as a result of their detachment from the microtubule-nucleating centres.

Ultraviolet (280 nm) microbeam irradiation of the centrosome (spindle pole) in the early anaphase slows down and then stops chromosome movement towards the irradiated pole. This happens as a result of rapid (in 1-2 min) disorganization of the half-spindle. Chromosome movement towards the opposite pole continues normally. Irradiation of the centrosome also affects cystotomy--the residual body is formed later than in the normal cell. In some cases additional constrictions are formed or the cytoplasm starts blebbing. Immediately after division the microtubule network in two daughter cells (one of them with irradiated centrosome) is similar. Two hours later in the irradiated cell the amount of microtubules is often less than in the sister cell. Incubation with nocodazole (0.5-1.5 h, 0.15 microgram/ml) shows that in the irradiated cells microtubules radiating from the centrosome are practically absent. Irradiation of other regions of the cytoplasm does not cause any of the effects described above.

Hybrids were produced between the Indian muntjak fibroblasts and rat Jensen sarcoma cell line (JF1) auxotrophic for asparagine. They were selected without cloning under conditions providing survival of parental Indian muntjak and hybrid cells. This allowed to compare the Indian muntjak chromosome variability in the parental cells and hybrids under identical culture conditions. The frequency of muntjak chromosome aberrations proved to de higher in the hybrids (up to 47%) than in the parental cells (6.5%). Predominant are chromosomal breaks and dicentrics. The latter are mainly formed by fusion of chromosomes 1 and 2. The most fragile are 1 and X-chromosomes. Chromosomal breaks are evenly distributed along chromosome 1, and "hot" points are observed in X-chromosome. Possible mechanisms of the Indian muntjak chromosome rearrangements induced by somatic cell hybridization are discussed.

Living cells of the monolayer cultures of embryonal pig kidney epithelium (PKE-cells) and of embryonal bovine tracheal cells (FBT-cells) were ultracentrifuged at 20,000g. The centrifugal force was directed parallel to the surface of the culture slides. Just after centrifugation, the cellular nuclei were displaced to the centrifugal parts of cells. Centrifuged slides with cells were returned to the normal culture conditions, and 22 h later the nuclei were seen to restore their central position in the cells. The motion of the nuclei to the cell center was rather chaotic both in direction and speed. The speed of this motion never exceeded several microns per hour. After nocodasole treatment (0.1-10 mkg/ml) of the cells or in a hypotonic medium, the distance of nuclear dislocation during centrifugation was longer, and the nuclei returned to the cell centers faster than in the control ones. After cytochalasin B treatment (2 mkg/ml), the nuclei moved to the cell centers somewhat more slowly than they did in the control cells. Thus, the establishment of the central position of nuclei in the cells takes place in the absence of microtubules or intermediate filaments. Probably, the central position of nuclei depends mainly on the action part of the cytoskeleton.

After administration of colcemid and 5-BrdU in the cell culture, the cells pass through the first interphase to delay in mitosis. Then the cells overcome the colcemid blockade, and polykaryocytes with micronuclei are formed. The second interphase in followed by the second mitosis, during which dicentric chromosomes are observed. These dicentrics are the result of telomeric chromosome fusion. The action of hyperthermia (40 degrees C) during the whole period of colcemid and 5-BrdU treatment or that of the hyperthermia (40 degrees C) only during the first 17 hours (the first interphase and the first mitosis) lead to the increased frequency of dicentrics. Under condition of hypothermia (34 degrees C) the frequency of dicentric formation decreases. Changes in cultivation temperature during the latest 25 hr of colcemid and 5-BrdU action (the second interphase and the second mitosis) exert no influence on the dicentric formation frequency. Because there are no dicentrics in cells during the metaphase of the first mitosis, it is supposed that the temperature--sensitive period may be the latest steps of colcemid blockade, i.e. the period of formation of micronuclei.

It has been found that irradiation in doses 0.5-2.0 Gy does not enhance the frequency of sister chromatid exchanges in cells of patients with Down's syndrome and ataxia-telangiectasia compared to the normal cells. In the case of ataxia, this phenomenon was accompanied with radioresistant replicative DNA synthesis, whereas in two cases of Down's syndrome the replicative DNA synthesis was found to be as radiosensitive as in the norm. According to these data, the mechanism of sister chromatid exchanges proposed in our previous publication (Pleskach et al., 1988) seems to be rather doubtful.

Polyanion (mannansulphate), cation dyes (Toluidine blue, Nile blue), anion-dye (Trypan blue) also non-electrolytes (urea, glutaraldehyde) were found to inhibit the phagosome-lysosome fusion in murine macrophages. The mechanism of this effect is discussed.

A method is proposed for experiment fulfillment and data analysis ("six classes" method) provided for proof or disproof of the reality of diverse influence of external factors on biological objects. The influence of alternating magnetic field on the mitotic index and chromosome aberration frequency in cultured diploid musculocutaneous cells of human embryos was estimated by this method. The revealed "plus-minus" effect did not correlate with the action of magnetic field but was caused by uncontrollable differences in conditions provided for various experiment replications.

The number of nucleoli was counted in the rabbit lymphocyte nuclei. This number was higher in meat rabbits than in skin-meat rabbits. We suppose that the polymorphism in the number of nucleoli reflecting the functional activity of ribosomal genes may be associated with biological-farming direction of productivity and energy of the animal growth.

The fine organization of the centromere structural heterochromatin (CSH) in a cell culture of calf trachea (TR) was studied by the methods of light and electron microscopy after fixation in native conditions and after treatments with water Henk's solutions and solutions of Ca2+ of different concentrations. In interphase nuclei the CSH forms compact blocks--the chromocentres, which are connected with the nuclear envelope or the nucleolus. The diameter of the main class of DNP fibers in the CSH, chromosomal arms and chromocentres after fixation of control cells and after treatment with 50% Henk's solution is 20-25 nm. 10-15 nm DNP fibers are largely found in the contacts with kinetochores. After 20% Henk's solution treatment 10-15 nm fibers predominate in the CSH and chromosomal arms. A chromonema--a 100 nm chromatin fiber--is detected in the CSH after treatments with different concentrations of Ca2+ solutions, as well after fixation in native conditions. The peculiarity of structural organization and properties of the calf CSH at interphase and mitosis as compared with analogous regions in mouse chromosomes are suggested to be connected with the composition of its DNA and proteins.

The monolayer culture of 3-day-old rat hepatocytes was treated by antimicrotubule agent colchicine after activation with isoproterenol. Alterations in the amount of lysosomes with the size of 1.0-2.5 and 3-6 mkm per cell were studied with light microscope after staining with the vital dye neutral red and after identification of acid phosphatase by plumbum precipitation in the frozen preparations. It was shown that isoproterenol application (20 microM) within 1.5 h did not change significantly the number of lysosomes sized 1.0-2.5 microns but stimulated the increase in the amount of lysosomes 3-6 mkm in size. The addition of 2.5 mM colchicine, after the isoproterenol treatment, decreases greatly the number of lysosomes with the size of 1.0-2.5 and 3-6 mkm. A considerable part of the large lysosomes can be resistant to the colchicine action. It is concluded that the microtubules play an important role in lysosome functioning.

A new method of cell fusion is proposed utilizing treatment with 15% solution of DMSO in serum before and after PEG treatment. With such treatments in SPEV cell culture a higher rate of cell fusion was obtained than that with other known methods of cell fusion. In the first wave of mitoses (0.5-4 h) mainly asynchronous division of nuclei, premature chromosome condensation and formation of telophase-like nuclei were observed in polykaryons. In the period of the second wave (14-20 h), mitoses were mainly synchronous and completed with cytokinesis. Micronuclei were formed frequently as a result of such mitoses. After the first wave of mitoses the number of polykaryons with pycnotic chromosomes sharply increased, and after the second wave of mitoses the number of polykaryons with pycnotic nuclei increased. The results obtained allow to conclude that heterophasic condition of the fused cells is one of the causes of pathological mitosis of polykaryons and of their death.

The blood of 6 newborn boys with haemolytic disease of newborns and of 3 healthy newborn boys was examined. In all 949 metaphase plates were analysed. In all the investigated plates the karyotype was masculine, 46,XY [correction of 46HY]. The mitotic index is lowered considerably in sick children that suggests inhibition of their cellular immunity. In these conditions, the K and L lymphocytes of donor blood, introduced in substituting blood transfusion, can interact with the sensitized erythrocytes in accordance with the type of reaction "the transplant against the host" causing the increase in the level of bilirubin after the operation. It is expedient to study the possibility of realization of substituting blood transfusion using the leucocyte-free blood.

A 5 second UV microirradiation of the centrosome during the early metaphase leads to a rapid (within 5 minutes) chromosome shift towards the normal pole and disorganizes the spindle. The later metaphase plate is also disorganized, chromosomes being situated chaotically in the central part of the cell. Numerous (up to 10 or more) microtubule convergence centers are observed instead of the spindle. 2-4 hours after the microirradiation some cells may enter cytotomy. The microirradiation of chromosomes and cytoplasm in similar and greater doses (exposure up to 15 seconds) did not lead to disorganization of the spindle and did not effect the normal completion of mitosis. Sometimes the 5 second microirradiation in the middle metaphase also blocked anaphase, but the microirradiation within the last 5 minutes of the metaphase always failed to block anaphase and normal completion of division.

After thawing cells, previously cryopreserved in the presence of dimethyl sulfoxide (DMSO), a decrease in their viability and increase in unscheduled DNA synthesis was observed. In 7 days, these parameters restored to the control level. Cryopreservation without DMSO resulted in the decrease in both cell viability and replicative and unscheduled DNA synthesis. In 14 days, these characteristics were seen to return to the normal level. Cryopreservation of cells without DMSO and their preservation in liquid nitrogen induced the frequency of chromosomal aberrations, mostly chromosomal breaks. The frequency of chromosomal aberrations increased with the duration of cell preservation in liquid nitrogen. The normal level was achieved following 7 days after cell thawing. Cells treated with DMSO only (without cryopreservation) display an increased number of chromosomal and chromatid breaks and translocations. Nonrandom distribution of chromosomal aberrations was observed, with particular chromosomes being involved in the appearance of dicentrics and translocations. The data obtained indicate that cryoprotective activity of DMSO is probably associated with the cell repair systems. The detected antimutagenic and mutagenic activity of DMSO may presumably reflect various conditions for its interaction with cells (with or without cryopreservation), as well as it may be specific for the muntjac cell line used in the present work.

Experimental data on the influence of culture conditions on the frequency of sister chromatid exchange (SCE) are reviewed. Evidence is provided that spontaneous SCEs exist. Various methods for the identification of spontaneous SCEs are discussed.

Formation of extracellular matrix structures in cultures of rat liver epithelial nontransformed cell line IAR2 was studied with antisera to fibronectin, laminin and type IV collagen by immunofluorescence and immunoelectron microscopy of platinum replicas. Fibronectin formed peripheral spots of variable size some of which outlined free cell edges, as well as fibrils located towards the center of single cells or of cellular islands. Similarly distributed structures were seen in isolated matrices. Codistribution of fibronectin and actin was observed only for the peripheral line of fibronectin spots and marginal circular actin bundle. Basement membrane components. laminin and type IV collagen, formed mainly spots of variable size predominantly beneath the cell or each cell in an island. Occasional fibrils were seen also. Essentially the same results were obtained by immunofluorescence and immunogold electron microscopy. Cytochalasin D treated cells displayed spots of both fibronectin and laminin. The relevance of previously postulated receptor-mediated assembly of extracellular matrix structures to the epithelial cells is discussed.

Cytochemical and molecular peculiarities of heterochromatic regions of bovine chromosomes have been studied, using specific fluorochrome staining induced decondensation, in situ hybridization, pretreatment of restriction enzymes. The heterochromatin of autosomes demonstrated a strong homogeneity. In chromosome Y two small specific heterochromatic regions were found lacking a long repeated tandem block of nucleotides enriched in GC base pairs and having no tandem block of Bkm repeats (10(4) b.p.). This class repeats are probably interspersed in the bovine genome. A rather seldom character of mammalian karyotypes is the absence of cytochemical heterochromatin in the X chromosome.

Karyological analysis of 6 lines with distinct tumorigenic properties of mouse strains C3H/He and C57BL/6 has been carried out using a differential staining of chromosomes. The number of normal copies of chromosomes varied in all the investigated cell lines. The more and the less stable chromosomes different from line to line. All the cell lines were characterized by decreased numbers of copies of normal chromosome 7; a decreased number of normal copies of chromosome 2 and 16 was detected in the course of the cell spontaneous neoplastic evolution. The decreased number of normal copies of chromosomes 8, 12 and X, and the increased number of normal copies of chromosome 10 were specific of the cell lines with intermediate tumorigenicity. The maximum tumorigenic cell lines differed from all other lines by increased numbers of copies of chromosomes 4 and 5, and by a decrease in copy number of chromosome 6. The data obtained are discussed in terms of the search of the regularity of karyotypic changes in the course of the cell neoplastic evolution.

The incubation of frog erythrocytes in the Ringer solution with novocaine (4.6 x 10(-3) M) during 24 hours at 10 degrees C provoked vacuole formation (segregation zones). Changes of the novocaine solution for a fresh Ringer solution and the following 48 hour incubation was accompanied by a decrease in the number of vacuoles both electron-translucent and containing membranous material. Simultaneously, the number of vacuoles with amorphous material only and with amorphous and membranous substances was seen to increase. Under the action of cycloheximide (1.10(-2) M) or oligomycin (2.5 x 10(-6) M) on erythrocytes with preformed vacuoles for 48 hours the total number of vacuoles and their dimensions decreased, with numerous amorphous inclusions appearing. Vacuoles with amorphous and membranous material increased in size. Similar ultrastructural changes in the segregation zones under the influence of both the inhibitors were observed showing the appearance of thick threads and a decreased share of electron-translucent vacuoles. A specific effect of cycloheximide, compared to that of oligomycin, involved the expansion of smooth endoplasmic reticulum cisternae. Under the influence of novocaine, 3H-leucin incorporation in proteins of frog erythrocytes was intensified. However, this incorporation was considerably inhibited by cycloheximide. Erythrocytes with segregation zones were more inhibitor susceptible than erythrocytes without vacuoles. The inhibitory effect was stronger early after their administration to the incubation medium, compared to the later periods.

The treatment of isolated metaphase chromosomes with 5 mM Tris buffer caused their decondensation into DNP fibers 10 nm in diameter. The following increase in CaCl2 concentration induced the transition of nucleosomic DNP fibers into DNP fibers 20 nM and 40-50 nM in diameter, and the recovery of the whole chromosomes. However, in the similar conditions, the typical chromosomes (threads about 100 nm thick), chromomeres and G-bands were not reconstructed. According to these data, we assume that DNP threads 40-50 nm in diameter may be artificial (i.e. "pseudochromonemes"). The treatment of isolated chromosomes with 0.35 and 0.6 M NaCl prevents from formation of nucleomeric and pseudochromomeric fibers, although bodies of chromosomes can be recovered after the removal of HMG and H1 proteins. These observations point to a high stability of chromosomal fasteners providing the structural integrity of mitotic chromosomes.

Under a long treatment of cells CHO-6 with 5-bromodeoxyuridine and colcemid it was discovered that dicentric chromosomes in metaphases of the first division of polykaryocytes contained micronuclei. When the cells were treated with colcemid only, the number of dicentrics was less. This phenomenon was described earlier in the Chinese hamster cell line B11d-ii FAF28. Under the similar testing of continuous cell lines of human, swine, African green monkey and Syrian hamster origin this result failed be reproduced. From the data of experiments it was testified that the phenomenon in question may be very likely the characteristics of all the Chinese hamster continuous cell lines.

The karyological analysis of the cell line 4647 used for production of a killed vaccine to Hepatitis A virus was run in the 98th, 107th, 117th and 127th passages by the routine and C, G, and Ag methods of differential chromosome staining. A considerable balancing of the chromosome composition at 107-127 passage levels is shown. The cells of line 4647 present a significant heterogeneity, as to the number of chromosomes, and do not belong to any distinct modal class. The modal number of chromosomes ranged from 61 to 66 and from 121 to 125 for hyperdiploid and polyploid cells, respectively. The stable modal class of cells was established in the tetrasomic region, when culturing in the medium containing 10% CS from the 107th passage, and in the medium containing 10% FBS from the 117th passage, which conforms to one of the WHO requirements asserted to the substrate cells.

Sister chromatid exchange (SCE) frequency at different times of fixation was studied in human lymphocyte cultures obtained from 6 donors. No differences were found in the SCE frequency between human lymphocyte cultures fixed at 72 and 96 hours of incubation (10.61 +/- 0.85 and 10.15 +/- 0.81 SCE per cell, respectively). However, a decreased SCE frequency (8.11 +/- 0.36 SCE per cell) was observed in cultures fixed at 120 hours of incubation. For a more detailed studies, one lymphocyte culture was fixed at different times of incubation (from 56 to 128 hours, at each a 8 hours). A slight increase in SCE frequencies was found at the interval between 56 and 88 hours of incubation, while starting from 104 hours of incubation a marked decrease in the SCE frequency was observed. Time-dependent changes in the SCE frequency may be described by the equation y = -1.8614 + 0.3922x - (2.5183 x 10(-3))x2, where y is the number of SCEs per cell, and x--the duration of culture incubation in hours. The observed phenomenon may be associated with changes in proportion of T and B lymphocytes, or with heterochromatization of chromosomes during a prolonged cultivation, or with an early in vitro stimulation of the in vivo long-lived lymphocytes that may be more damaged than the in vivo short-lived and the in vitro late-stimulating ones.

Levels of spontaneous and of C-mitomycine and cyclophosphane-induced chromosome aberrations are determined among the persons not subject to professionally industrial hazards. It has been revealed that in all the experimental series the aberration rate in those, who carried extreme polymorphic variants, was much higher compared to those without these variants in their karyotypes. Among the persons with the above extreme versions, the impact of chromosome ruptures is stronger in the vicinity of the structural heterochromatin. Possible causes of the discovered phenomenon are discussed.

The influence of polyamines on the phagosome-lysosome fusion in murine peritoneal macrophages and on polymerization of G-actin from the rabbit muscle in vitro has been studied. Both natural polyamines (spermin, spermidin, putrescin) and synthetic phenyl derivates of polyamines (3,3'-diaminobensidin, 1,5-naphtalin diamine, 4,4'-diaminodiphenilmetan, dancylcadaverin) were used. Unlike the phenyl derivates of polyamines and putrescin, spermin and spermidin stimulate the phagosome-lysosome fusion to induce G-actin polymerization. Possible mechanisms of action of the above polyamines are discussed.

Lysosomal inhibitors (amino acid methyl esters) and the platelet-derived growth factor stimulate the resting NIH 3T3 cells to enter the S period. Incubation of cells in the medium containing lysosomal inhibitors enhances protein accumulation and seemingly does not disrupt lysosomes. These results indicate that proliferative homeostasis depends on the metabolic status of cells and that catabolic processes activated in resting cells negatively influence the prereplicative reactions.

Sister chromatid exchanges (SCE) and cellular proliferation were studied in lymphocytes from 16 pregnant and 18 non-pregnant women. A lowered SCE frequency was found in lymphocytes obtained from pregnant women (9.41 +/- 0.39 vs. 11.07 +/- 0.42 SCE/metaphase in non-pregnant women). Lower proliferation rates were also common for cultures of pregnant women. Thus, physiological changes occurred in the organism of pregnant women may influence various cytogenetic indices registered in human peripheral blood cultures.

The mitochondrial complex condition of continuous CEMT4 cell line infected by the human immunodeficiency virus has been investigated. The mitochondrial morphology of these and of intact cells was similar in great extent, though several changes were observed. For example, mitochondrial profiles with multiple dichotomous branches and anastomosis cristae were noted in the former. These changes resulted in the augmentation of the inner membrane square of mitochondrion. The formation of mitochondrial clusters connected with special junctions was a very characteristic part of the infected cell. Contacts were seen to be formed between the outer membranes neighboring profiles. These contacts look as X-like little bridges, or net-like or plate-like structures. The mutual transition of all these structures was observed using goniometer adapter. As has been shown by the three-dimensional reconstruction of mitochondrial junction zones, this area is presented by a single mitochondrion being structurally very complicated and very large in size compared to the neighbouring ones.

Cytoskeleton organization of cultured normal epithelial cells (epithelium of newborn mouse kidney, mouse and rat hepatocytes) was studied using electron microscopy of platinum replicas. These cells in culture were firmly connected with each other and formed multicellular islands. Pseudopodial activity was observed only at the free edges of marginal cells of the islands. Cytoskeleton in the vicinity of such active edges included several structurally different zones. The most peripheral zone contained dense actin meshwork. More inner "sparse" zone contained loose actin filament network. Next zone in the same direction was the lamella proper. It contained individual microfilaments and their bundles or meshwork patches. Microtubules and intermediate filaments were also present in the lamella proper. The characteristic feature of the central (endoplasmic) region of the marginal cells of the islands was the presence of the submembranous microfilament sheath. Microfilaments in the sheath were densely packed. Individual fibers were visible along a significant distance. The inner cells in the epithelial islands had no zonal organization of the cytoskeleton. The endoplasmic microfilament sheath occupied the whole dorsal cell surface in these cells. Different epithelia studied here had some variations in the relative width of cytoskeletal zones. The organization of cytoskeleton in the epithelial cells has many features in common with that in fibroblasts. Possible mechanisms of establishment of the zonal cytoskeletal organization in both the cell types are discussed.